Biomimetic evolutionary analysis: testing the adaptive value of vertebrate tail stiffness in autonomous swimming robots.

For early vertebrates, a long-standing hypothesis is that vertebrae evolved as a locomotor adaptation, stiffening the body axis and enhancing swimming performance. While supported by biomechanical data, this hypothesis has not been tested using an evolutionary approach. We did so by extending biomimetic evolutionary analysis (BEA), which builds physical simulations of extinct systems, to include use of autonomous robots as proxies of early vertebrates competing in a forage navigation task. Modeled after free-swimming larvae of sea squirts (Chordata, Urochordata), three robotic tadpoles (;Tadros'), each with a propulsive tail bearing a biomimetic notochord of variable spring stiffness, k (N m(-1)), searched for, oriented to, and orbited in two dimensions around a light source. Within each of ten generations, we selected for increased swimming speed, U (m s(-1)) and decreased time to the light source, t (s), average distance from the source, R (m) and wobble maneuvering, W (rad s(-2)). In software simulation, we coded two quantitative trait loci (QTL) that determine k: bending modulus, E (Nm(-2)) and length, L (m). Both QTL were mutated during replication, independently assorted during meiosis and, as haploid gametes, entered into the gene pool in proportion to parental fitness. After random mating created three new diploid genotypes, we fabricated three new offspring tails. In the presence of both selection and chance events (mutation, genetic drift), the phenotypic means of this small population evolved. The classic hypothesis was supported in that k was positively correlated (r(2)=0.40) with navigational prowess, NP, the dimensionless ratio of U to the product of R, t and W. However, the plausible adaptive scenario, even in this simplified system, is more complex, since the remaining variance in NP was correlated with the residuals of R and U taken with respect to k, suggesting that changes in k alone are insufficient to explain the evolution of NP.

Low stomatal and internal conductance to CO2 versus Rubisco deactivation as determinants of the photosynthetic decline of ageing evergreen leaves.

A novel A-Ci curve (net CO2 assimilation rate of a leaf -An- as a function of its intercellular CO2 concentration -Ci) analysis method (Plant, Cell & Environment 27, 137-153, 2004) was used to estimate the CO2 transfer conductance (gi) and the maximal carboxylation (Vcmax) and electron transport (Jmax) potentials of ageing, non-senescing Pseudotsuga menziesii leaves in relation to their nitrogen (N) content and protein and pigment composition. Both gi and the stomatal conductance (gsc) of leaves were closely coupled to Vcmax, Jmax and An with all variables decreasing with increasing leaf age. Consequently, both Ci and Cc (chloroplastic CO2 concentration) remained largely conserved through successive growing seasons. The N content of leaves, as well as the amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and other sodium dodecyl sulfate-soluble proteins, increased during the first three growing seasons, then stabilized or decreased only slightly afterwards. Thus, the age-related photosynthetic nitrogen use efficiency (PNUE) decline of leaves was not a consequence of decreased allocation of N towards Rubisco and other proteins involved in bioenergetics and light harvesting. Rather, loss of photosynthetic capacity was the result of the decreased activation state of Rubisco and proportional down-regulation of electron transport towards the photosynthetic carbon reduction (PCR) and photorespiratory (PCO) cycles in response to a reduction of CO2 supply to the chloroplasts' stroma. This study emphasizes the regulatory potential and homeostaticity of Cc- rather than photosynthetic metabolites or Ci- in relation to the commonly observed correlation between photosynthesis and gsc.

Water stress decreases the transfer conductance of Douglas-fir (Pseudotsuga menziesii) seedlings.

We tested the hypothesis that transfer conductance (gi) of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings is reduced by water stress. Seedlings were irrigated with a solution of 25% polyethylene glycol so as to impose water stress rapidly, thereby limiting acclimatory responses. Transfer conductance was measured pre-treatment and post-treatment by two methods. Water stress reduced net photosynthesis by 20-50%. The initial slope of the rate of photosynthesis (A) over the intercellular carbon dioxide (CO2) concentration (Ci) response was reduced by water stress, indicating that reduced photosynthesis was not wholly accounted for by reduced stomatal conductance. The carbon isotope and chlorophyll fluorescence methods both indicated that water stress decreased gi. From isotopic measurements with 1% O2, gi was 0.076 +/- 0.009 (mean +/- SE) mol m(-2) s(-1) in well-watered seedlings and 0.044 +/- 0.004 mol m(-2) s(-1) in water-stressed seedlings. Fluorescence estimates of gi were 0.08 +/- 0.01 mol m(-2) s(-1) in well-watered seedlings and 0.044 +/- 0.004 mol m(-2) s(-1) in water-stressed seedlings. The drought-induced reduction in gi was responsible for the reduction in slope of the A/Ci response, and thus there was no difference in the slope of the A over the chloroplastic CO2 concentration (Cc) response between treatments and no indication of impaired mesophyll metabolism. These data illustrate that impairments of mesophyll metabolism can be revealed only from analysis of the A/Cc response.

Photosynthetic responses and N allocation in Douglas-fir needles following a brief pulse of nutrients.

The temporal distribution of soil nutrients is heterogeneous, and thus the uptake, storage and later remobilization of brief nutrient pulses may be critical for growth in nutrient-limited habitats. We investigated the response of photosynthesis and the major nitrogen (N) fractions in needles of 2-year-old Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings to a 15-day nutrient pulse (containing 250 ppm N). The nutrient pulse (N pulse) was imposed in late July, toward the end of the seedlings' third growing season, and subsequent changes in photosynthesis and needle N fractions were examined over the following 3 months. Needles are sites of photosynthesis and putative storage organs. Thus we tested two hypotheses: (1) N from the N pulse is quickly synthesized from soluble non-protein N into soluble proteins, especially Rubisco, and (2) the N pulse increases photosynthetic rates and thus growth. We also examined an alternative hypothesis that Rubisco functions also as a storage protein, in which case we would predict increases in amount of Rubisco in response to the N pulse without concomitant increases in photosynthesis. Soluble non-protein N was the most dynamic N pool and may have constituted a temporary storage reservoir; however, the quantitative significance of soluble non-protein N is questionable because this pool was at most only 7% of total N. Concentrations of Rubisco were unaffected by the N-pulse treatment and there was little evidence that Rubisco served as a storage protein. Nutrient-pulse seedlings added twice as much dry mass as controls during the 3 months post-treatment (Warren et al. 2003a). Over the same period, the maximum rate of light-saturated photosynthesis (A(max)) declined to low rates in control seedlings, whereas A(max) increased in N-pulse seedlings. Nevertheless, treatment and temporal trends in N and Rubisco content per unit area were poorly related to A(max), and it seems likely that photosynthesis was limited by additional factors, perhaps thylakoid proteins or an inadequate supply of other nutrients.

Response of Douglas-fir seedlings to a brief pulse of 15N-labeled nutrients.

The temporal distribution of soil nutrients is heterogeneous, and thus the uptake, storage and later remobilization of brief nutrient pulses may be critical for growth in nutrient-limited habitats. We investigated the response of 2-year-old Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings receiving a low nutrient supply to a 15-day nutrient pulse (containing 250 ppm nitrogen (N) as 10 atom % 15NH4 15NO3). The nutrient pulse was imposed in late July, toward the end of the seedlings' third growing season, and subsequent changes in dry mass and N content over the following 3 months were determined from destructive harvests. We tested three hypotheses: (1) N from the nutrient pulse is rapidly assimilated and accumulated primarily in needles and roots; (2) this accumulated N is later remobilized to support new growth; and (3) the nutrient pulse leads to a larger second flush of shoot growth. Seedlings increased their N content by 175 mg (67%) in response to the nutrient pulse. Nitrogen was taken up preferentially into younger tissues, especially the secondary flush and current-year roots. Immediately after the nutrient pulse, tissue N concentrations were high and supported subsequent increases in dry mass. Over 3 months, seedlings receiving the nutrient pulse added twice as much dry mass as control seedlings, and even after 3 months of growth, N concentrations remained greater than in controls. Current-year and older needles were the only components whose dry mass did not increase over this period. The nutrient pulse increased the size of the second flush, but it was still a minor component of increments in dry mass (approximately 10% of the total dry mass increment) and N content (23%). The relatively modest increases in N content during autumn could be accounted for by soil uptake and there was no evidence that N was remobilized to support growth of new tissues. Short-term (15 days) elevated N uptake led to sustained growth in the long term (> 3 months), and thus growth rate was to a large extent decoupled from current nutrient supply.

Responses of gas exchange to reversible changes in whole-plant transpiration rate in two conifer species.

This study examined the autonomy of branches with respect to the control of transpiration (E) in Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) and western red cedar (Thuja plicata Donn) seedlings. Experiments were conducted on whole seedlings in a gas exchange system with a dual-cuvette that permitted independent manipulation and measurement of E in the upper and lower cuvettes. The value of E in one cuvette was manipulated by varying vapor pressure deficit (D) between 2.2 and 0.2 kPa, whereas D in the other cuvette was held at 2.2 kPa. Reducing D, while increasing stomatal conductance (gs), resulted in an overall decrease in E. In western red cedar, this decrease was almost threefold, and in Douglas-fir, approximately fourfold. In well-watered western red cedar, a reduction of whole-plant E by 46% (brought about by reducing D in the upper cuvette) resulted in a 12% increase in gs, a 12% increase in E and a 7% increase in net assimilation (A) of untreated foliage in the lower cuvette. Responses of gs, E and A of untreated foliage were similar irrespective of whether foliage was at the top or bottom of the seedling. When D in the treatment cuvette was restored to 2.2 kPa, gs, E and A of foliage in the untreated cuvette returned to pretreatment values. In contrast, in well-watered Douglas-fir, there was almost no change in gs, E or A of untreated foliage in one cuvette when D in the other cuvette was reduced, causing a 52% reduction in whole-plant E. However, similar manipulations on drought-stressed Douglas-fir led to 7-19% increases in gs, E and A of untreated foliage. In well-watered western red cedar, daytime leaf water potential (Psil) was maintained near -0.9 MPa over a wide range of D, whereas Psil of Douglas-fir decreased from -1.2 to -1.5 MPa as D increased. The tighter (isohydric) regulation of Psil in western red cedar may partly explain its greater stomatal response to D and variation in whole-plant E compared with Douglas-fir. In response to a reduction in E, measured increases in Psil and gs of unmanipulated foliage were less than predicted by a model assuming complete hydraulic connectivity of foliage. Our results suggest the foliage of both species is partially autonomous with respect to water.

Responses of transpiration and photosynthesis to reversible changes in photosynthetic foliage area in western red cedar (Thuja plicata) seedlings.

Experiments were conducted on 1-year-old western red cedar (Thuja plicata Donn.) seedlings to determine the response of illuminated foliage to reversible changes in total photosynthetic foliage area (L(A)). Reductions in L(A) were brought about by either shading the lower foliage or by reducing the ambient CO2 concentration (c(a)) of the air surrounding the lower part of the seedling. In the latter case, the vapor pressure was also changed so that transpiration rates (E) could be manipulated independently of photosynthetic rates (A). We hypothesized that following such treatments, short-term compensatory changes would occur in stomatal conductance (g(s)) and A of the remaining foliage. These changes would occur in response to hydraulic signals generated by changes in the water potential gradient rather than changes in the distribution of sources and sinks of carbon within the seedling. When a portion of the foliage was shaded, there was an immediate reduction in whole-seedling E and a concomitant increase in g(s), A and E in the remaining illuminated foliage. However, the intercellular CO2 concentration did not change. These compensatory effects were fully reversed after the shade was removed. When the lower foliage A was reduced to < 0 micromol m-2 s-1, by shading or lowering c(a), and E was either unchanged or increased (by adjusting the vapor pressure deficit), there was no significant increase in g(s) and A in the remaining foliage. We conclude that compensatory responses in illuminated foliage occur only when reductions in L(A) are accompanied by a reduction in whole-plant E. The relationship between the reduction in whole-seedling E and the increase in A is highly linear (r2 = 0.68) and confirms our hypothesis of the strong regulation of g(s) by hydraulic signals generated within the seedling. We suggest that the mechanism of the compensatory effects is a combination of both increased CO2 supply, resulting from increased g(s), and a response of the rate of carboxylation, possibly related to the activity of Rubisco.

Desiccation, cryopreservation and water relations parameters of white spruce (Picea glauca) and interior spruce (Picea glauca x engelmannii complex) somatic embryos.

Effects of drying and cryopreservation on survival of spruce (Picea glauca (Moench) Voss and Picea glauca x engelmannii complex) somatic embryos (SEs) were investigated with the aim of developing simple and robust protocols for embryo storage. Somatic embryos dried over salt solutions of known water potential (Psi) survived removal of virtually all free water, to a relative water content (RWC) of approximately 0.13, a value similar to that for spruce zygotic embryos from dry seed. Desiccated SEs also survived subsequent freezing in liquid nitrogen, without the addition of cryoprotectant or pre-culture steps. Highest survival (> 80%) after freezing in liquid nitrogen was in embryos pre-dried to Psi of -15 to -20 MPa, which yielded RWC close to predicted bound (apoplastic) water values. Low (< 35%) or no survival after freezing was observed in embryos pretreated at higher Psi (above -5 MPa) or at very low Psi (-540 MPa, using silica gel), respectively.

Plasticity in water-use efficiency of Picea sitchensis, P. glauca and their natural hybrids.

Plasticity in water-use efficiency (WUE) was examined in populations of Picea glauca (Moench) Voss and P. sitchensis (Bong.) Carr. and their natural hybrids from an ecocline along the Skeena Valley, British Columbia, which runs from the dry continental interior (P. glauca) to the wet maritime Pacific coast (P. sitchensis). Seedlings were grown in a growth chamber and kept well-watered or repeatedly droughted for 3 months. Mean population photosynthetic WUE and total tissue δ(13)C values were strongly correlated within and across treatments (r=0.95). There were also strong correlations (r=0.60-0.80) between individual seedling total tissue δ(13)C and dry mass, and δ(13)C and net photosynthesis (A), indicating that variation in A was primarily responsible for differences in δ(13)C. When kept well-watered, P. sitchensis and the hybrids had higher δ(13)C (-27.8‰ and -27.5‰, respectively) and higher dry mass (2.17 g and 1.99 g, respectively) than P. glauca (-28.2‰ and 1.68 g). Species ranking was reversed by repeated drought, with P. glauca and the hybrids having higher δ(13)C (-25.6‰ and -25.5‰, respectively) and dry mass (1.10 g and 1.08 g, respectively) than P. sitchensis (-26.4‰ and 0.98 g). P. glauca had a smaller decrease in dry mass (35%) and a bigger increase in δ(13)C (by 2.7‰) than P. sitchensis (55% and 1.4‰, respectively), with the hybrids in between (45% and 2.0‰, respectively). Drought also had a greater effect on A in P. sitchensis (36% reduction) than in P. glauca (14% reduction) or the hybrids (24% reduction). Thus P. glauca and, to a lesser extent, hybrid populations, performed better and were more plastic than P. sitchensis in response to water deficit. Under the well-watered treatment, the hybrids behaved more like P. sitchensis in growth and WUE. These patterns are consistent with the seasonal variation in moisture availability that occurs along the introgression zone.

Effects of injectate volume on thermodilution measurements of cardiac output in patients with low ventricular ejection fraction.

OBJECTIVE: To determine the effect of 5-mL injectate on cardiac output measurements in critically ill patients with low ventricular ejection fraction (< 35%). METHODS: Thermodilution cardiac output measurements obtained with three 5-mL and three 10-mL (randomly ordered) iced injectates in 50 patients with low ejection fraction were averaged if the measurements were within 10% of the median. If the 3 measurements were not within those limits, additional measurements were obtained. RESULTS: Cardiac output measured with the 5-mL injectate (mean, 4.63 L/min) and cardiac output measured with the 10-mL injectate (mean, 4.52 L/min) were not significantly different (P = .64). Lower and upper limits of agreement were -1.7 L/min to +1.6 L/min. The bias (mean difference between 10- and 5-mL measurements) of all measurements was -0.09, and the precision was 1.43 L/min, with a 95% confidence limit (mean difference +/- 2 SD) of -1.7 to +1.6 L/min. An additional measurement was necessary in 77% of patients in the 5-mL group but in only 48% of the 10-mL group (P = .006). CONCLUSIONS: Cardiac outputs measured with 5- and 10-mL injectates do not differ significantly. The greater variability of measurements obtained with a 5-mL injectate suggests that more measurements, and thus more time, are needed to measure cardiac output accurately. Clinicians must weigh the benefit of minimizing fluid volume used against the potential decreased reliability of cardiac output measurements.

Impact of a comprehensive heart failure management program on hospital readmission and functional status of patients with advanced heart failure.

OBJECTIVES: To assess the impact of a comprehensive heart failure management program, functional status, hospital readmission rate and estimated hospital costs were determined and compared for the 6 months before and the 6 months after referral. BACKGROUND: The course of advanced heart failure is characterized by progressive clinical deterioration reflected in frequent hospital admissions, which comprise the major financial cost. METHODS: Over a 3-year period, 214 patients were accepted for heart transplantation and discharged after evaluation, which included adjustments in medical therapy and intensive patient education. Patients were in New York Heart Association functional class III or IV (94 and 120 patients, respectively), with a mean left ventricular ejection fraction of 0.21, peak oxygen consumption of 11 ml/kg per min and a total of 429 hospital admissions in the previous 6 months (average 2.0 per patient). Changes in the medical regimen included a 98% increase in angiotensin-converting enzyme inhibitor dose and a flexible diuretic regimen after 4.2-liter net diuresis, with counseling also regarding diet and progressive exercise. RESULTS: During the 6 months after referral, there were only 63 hospital readmissions (85% reduction), with 0.29/patient (p < 0.0001). Functional status improved as assessed by functional class (p < 0.0001) and peak oxygen consumption (15.2 vs. 11.0 ml/kg per min, p < 0.001). The same results were seen after excluding the 35 patients without full 6-month follow-up (9 deaths, 14 urgent transplant procedures during hospital readmission, 12 elective transplant procedures from home); 34 hospital admissions occurred after referral, compared with 344 before referral. Even when adding in the initial hospital admission after referral for these 179 patients, there was a 35% decrease in total hospital admissions in the 6-month period. The estimated savings in hospital readmission costs after subtracting the initial hospital costs for management was $9,800 per patient. CONCLUSIONS: Comprehensive heart failure management led to improved functional status and an 85% decrease in the hospital admission rate for transplant candidates discharged after evaluation. The potential to reduce both symptoms and costs suggests that referral to a heart failure program may be appropriate not only for potential heart transplantation, but also for medical management of persistent functional class III and IV heart failure.

A whole-plant cuvette system to measure short-term responses of conifer seedlings to environmental change.

A computer-controlled whole-plant cuvette system is described that allows precise and independent control of temperature (+/- 0.05 degrees C), vapor pressure (+/- 0.02 kPa), CO(2) concentration (+/- 2 micro mol mol(-1)) and photosynthetic photon flux density (+/- 5 micro mol m(-2) s(-1)), and allows the continuous measurement of net photosynthesis and transpiration rates. Vapor pressure is controlled by circulating chamber air through a CaSO(4) desiccant column supported on a digital balance. Transpiration rate is calculated from the change in desiccant mass with time. Photosynthesis rate is measured by integrating the output of a mass flow controller used to inject CO(2) into the chamber to compensate for that assimilated by the plant. The control system can be driven by set points that can be varied, for example, as a function of time, or held constant. We were able to simulate weather data obtained from climate stations and accurately follow, in real time, the output of sensors measuring outside conditions. Experiments on well-watered one- and two-year-old nursery-raised western red cedar (Thuja plicata Donn.) and white spruce (Picea glauca (Moench) Voss) seedlings showed that if the mean daily temperature was increased from 20 to 22 degrees C with vapor pressure remaining constant at 1 kPa, CO(2) concentrations must almost double to compensate for the decrease in net photosynthesis rate.

Water relation parameters of embryogenic cultures and seedlings of larch.

Changes in the water relations parameters of developing somatic embryogenic and xygotic European larch (Larix decidua) were studied. Water release curves were generated by suspending tissue samples over unsaturated NaCl solutions until they reached vapor equilibration with the surrounding air. Twenty solutions were used whose water potentials ranged from -0.05 to -10 MPa. Water release curves were obtained by plotting paired values of tissue relative water content (RWC) and solution potential. Curves were derived for embryonic larch at various stages of development and for hypocotyls and roots from germinated zygotic and somatic embryos. The ability to resist dehydration increased markedly with development. Stage 1 tissue, which consisted of clusters of loosely associated nonchlorophyllous cells, had extremely low bulk elastic modulus (epsilon) (1.91 MPa) and apoplastic water content (A) (0.023), relatively high osmotic potential (Psi(pi)) (-0.53 MPa), and lost turgor at 0.56 RWC. In contrast, mature embryoids with primary roots, hypocotyl, and cotyledons (stage 3) had an almost 4-fold increase in A (0.089), significantly higher epsilon (3.49 MPa), and lower Psi(pi) (-0.88 MPa) and lost turgor at 0.66 RWC. Hypocotyl tissue from germinated somatic embryos lost turgor at 0.74 RWC and had higher epsilon, A, and solute accumulation than pregerminated tissue. Hypocotyl tissue resisted dehydration more strongly than root tissue, and differences between root and hypocotyl water relation parameters were more pronounced in xygotic than in somatic seedlings. Highest dehydration resistance was in zygotic hypocotyls. The characterization of the water relations of tissue cultures should allow the development of more consistent and reliable desiccation protocols to induce maturation of embryos and produce synchronously germinating seed.

Calcitropic hormone levels in polynesians: evidence against their role in interracial differences in bone mass.

Blacks are known to have a higher bone mass than whites and have recently been found to have significantly different levels of calcitropic hormones and other biochemical indices of calcium metabolism. To assess the possible significance of these biochemical differences to interracial differences in bone mass, we have undertaken an assessment of indices of calcium metabolism in Polynesian subjects, since they also have a higher bone mass than whites. Serum concentrations of 25-hydroxyvitamin D were slightly lower in Polynesians than in whites (65 +/- 5 vs. 95 +/- 10 nmol/L; P less than 0.02), but there were no differences between the groups in serum levels of calcium (total and ionized), phosphate, magnesium, 1,25-dihydroxyvitamin D, PTH, calcitonin, alkaline phosphatase activity, and bone gla-protein. Furthermore, urinary excretion of hydroxyproline, calcium, phosphate, magnesium, sodium, and potassium and the tubular maximum for the reabsorption of phosphate were not different between whites and Polynesians. Intestinal strontium absorption was similar in the two groups. In contrast, distal forearm bone mineral content was higher in Polynesians (P less than 0.01) and midupper arm muscle area was also increased in this group (P less than 0.005). It is concluded that the higher bone mass of Polynesians cannot be attributed to alterations in the basal levels of calcitropic hormones, but may be related to their greater muscle mass. It is probable that the previously observed black-white differences in the vitamin D endocrine system are secondary to the effects of skin color on vitamin D synthesis and are not contributory to the lower bone mass of whites.

Characterisation of insulin-like growth factor I receptor in skeletal muscles of normal and insulin resistant subjects.

The insulin-like growth factor I receptor and the activity of its associated tyrosine kinase activity were characterised in wheat germ agglutinin extracts from skeletal muscle biopsies from nine control and ten obese Type 2 (non-insulin-dependent) diabetic subjects, who had marked peripheral in vivo insulin resistance for glucose disposal and hyperinsulinaemia. In parallel studies, the concentration of the insulin receptor and its tyrosine kinase activity were examined in the biopsy extracts and compared to the findings for the insulin-like growth factor I receptor system. Specific binding sites for insulin-like growth factor I were detected. The receptor binding of insulin-like growth factor I was not changed in the obese diabetic subjects as compared to binding activity in the biopsies from the control subjects. The molecular weight of the insulin-like growth factor I receptor alpha subunit was similar in both groups (135 kDa). The insulin-like growth factor I stimulated tyrosine kinase activity was also similar for the two groups. In contrast, insulin binding activity was 30% less in the receptor extracts from the in vivo insulin resistant group when compared to the control group. Moreover, insulin-stimulated tyrosine kinase activity was reduced in the former group by 40% when the value was corrected for insulin binding. Thus, specific insulin-like growth factor I receptors are present in human skeletal muscle. These receptors are normal in insulin resistant obese Type 2 diabetic subjects.(ABSTRACT TRUNCATED AT 250 WORDS)